P. Olivier — 1/2000
Solutions
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0.08% w/v Pepsin
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Dissolve 0.4 g pepsin in 500 mL of 0.1M HCl (496 ml dd H20 + 4.1 mL conc. HCl), filter, store 4ºC.
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2M HCl
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For 417 mL ddH20 plus 83 mL conc. HCl. Filter, store RT.
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0.1M Na Borate
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For 500 mL, 19.07 g Na borate, filter, store RT.
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IFA
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10mM HEPES, pH 7.4; 150mM NaCl; 4% fetal bovine serum; 0.1% sodium azide. Filter, store RT.
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IFA/Tween 20
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Add 0.5% Tween 20 to IFA.
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Vortexing pellets while adding solutions is important.
Fixation of Cells
- Trypsinize cells. Resuspend in PBS containing 5% serum (filtered to remove serum precipitate). Break up clumps by pipetting and spin down cells in 15 mL conical tubes.
- Aspirate supe and vortex pellet. While vortexing, add 1.5 mL of cold PBS. While vortexing, slowly add 3 mL of cold 95% ethanol in a steady stream. Continue to vortex until thoroughly mixed. At this point samples may be stored at 4ºC for several days or longer.
Staining Procedure
Spins are done in a clinical tabletop centrifuge 5' at top speed, approx 1000 X g
- Spin down cells, aspirate supe and vortex pellet.
- While vortexing, add 3 mL of 0.08% pepsin. Incubate at 37°C for 20 min with occasional mixing.
- Spin down nuclei (expect a very small pellet), aspirate supe and vortex pellet. While vortexing add 1.5 mL of 2M HCl. Incubate 20 min at 37°C with occasional mixing.
- While vortexing add 3 mL 0.1M Na-borate. Spin down nuclei.
- Aspirate supe and vortex pellet. While vortexing add 2 mL IFA/Tween 20.
- Spin down nuclei, aspirate supernatant and vortex pellet. Add 75 µL of 1:5 dilution in IFA of anti-BrdU-FITC and incubate on ice in the dark for 30 min.
- Add 2 mL IFA/Tween 20 while vortexing. Spin down nuclei, aspirate supe and vortex pellet. Resuspend in 0.25 mL IFA.
- Add 0.25 mL of 100 µg/mL propidium iodide (in PBS) and incubate on ice in the dark for 60 min. Samples can be stored overnight (even a day or so is OK).
- Run on FACScan.
Tags: Fero-Flow, Fero-Lab-Protocols
